1. Field of the Invention
The present invention relates to an isogenic strain line of a bacterium for producing polyhydroxyalkanoate in which a gene encoding polyhydroxyalkanoate synthase is disrupted; a gene targeting vector for disrupting a gene encoding polyhydroxyalkanoate synthase of a bacterium for producing polyhydroxyalkanoate; and a process for disrupting a gene encoding polyhydroxyalkanoate synthase of a bacterium for producing polyhydroxyalkanoate, using the aforementioned gene targeting vector. The present invention also relates to a method for producing polyhydroxyalkanoate comprising expressing a recombinant polyhydroxyalkanoate synthase in an isogenic strain line of a bacterium for producing polyhydroxyalkanoate in which a gene encoding polyhydroxyalkanoate synthase is disrupted.
2. Related Background Art
Until now, it has been reported that many microbes produce and accumulate in the body poly-3-hydroxy butyrate (PHB) or other poly-3-hydroxyalkanoate (PHA) (“Biodegradable plastic handbook”, edited by the biodegradable plastic study group, NTS Inc. P178-197 (1995)). These polymers, like conventional plastics, can be used for producing various products by melt processing and the like. Furthermore, these polymers have an advantage of being completely degraded by microbes in the nature and do not cause pollution by remaining in the natural environment, unlike many conventional synthetic polymers, because they are biodegradable. They are also superior in biocompatibility, and would be expected to have applications as soft material for medical use and the like. Recently in particular, it is expected that unusual PHA in which substituent groups other than alkyl group are introduced in the side chain would be very useful considering expanding application of microbially produced PHA, for example an application as a functional polymer. Examples of such substituent groups include groups containing an aromatic ring (phenyl group, phenoxy group, benzoyl group and the like), unsaturated hydrocarbons, ester group, aryl group, cyano group, halogenated hydrocarbons, epoxides, thioethers and the like.
It has been known that microbially produced PHA can have various compositions and structures, depending upon the species of microbes for use in production thereof, the composition of the medium, the culture condition and the like. Various researches have been carried out on such PHA producing microbes, and the biosynthetic pathway of PHA has been relatively well investigated. Up until now, polyhydroxyalkanoate synthase is classified into three classes by substrate specificity and subunit composition.
Polyhydroxyalkanoate synthase which belongs to “the first class” is found in Ralstonia eutropha, Aeromonas punctata and the like and uses, as a substrate, thioester conjugate of 3-hydroxyalkanoate with short carbon chain length of C3-C5 and coenzyme CoA. Polyhydroxyalkanoate synthase in this class is composed of a single subunit of molecular weight 61-73 kDa.
Polyhydroxyalkanoate synthase which belongs to “the second class” is found in Pseudomonas oleovolan and Pseudomonas aeruginosa and uses, as a substrate, thioester conjugate of 3-hydroxyalkanoate with medium carbon chain length of C6-C14 and coenzyme CoA. Polyhydroxyalkanoate synthase in this class is composed of a single subunit of molecular weight 61-73 kDa, and in general there are two genes (phaC1 and phaC2) of Polyhydroxyalkanoate synthase, forming, together with the polyhydroxyalkanoate depolymerase gene (phaZ), a cluster of phaC1-phaZ-phaC2.
Polyhydroxyalkanoate synthase which belongs to “the third class” is found in Allochromatium vinosum and Ectothiorhodospira shaposhnikovii and the like, and the substrate specificity is similar to that of the first class polyhydroxyalkanoate synthase and uses thioester conjugate of 3-hydroxyalkanoate with short carbon chain length of C3-C5 and coenzyme CoA. Polyhydroxyalkanoate synthase that belongs to this class is composed of 2 different kinds of subunits of about 40 kDa.
Now, targeting the improvement of PHA productivity and the development of microbes capable of producing novel PHA, studies are carried out to modify polyhydroxyalkanoate synthase using the evolutionary engineering approach. In APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 68,2411(2002), an evolutionary engineering modification was applied to the Aeromonas caviae derived polyhydroxyalkanoate synthase gene, which belonged to “the first class”, and Escherichia coli was transformed with this gene together with the genes of (R)-specific enoyl-CoA hydratase (phaJ) and granuleassociated protein (ohaP), and the transformants were screened. As the result, it was disclosed that the productivity of PHA, which was composed of random copolymerization of 3-hydroxy butyric acid (PHB) and 3-hydroxyhexanoic acid, was improved and the unit ratio of 3-hydroxyhexanoic acid was increased. Also, in Applied Microbiology and Biotechnology, 59.477 (2002), random mutations were introduced to the Aeromonas punctata derived polyhydroxyalkanoate synthase gene, which belongs to “the first class”, and Escherichia coli was transformed with this gene together with the genes of β-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB) derived from Ralstonia eutropha, and the transformants were screened. As the result, it was disclosed that mutated enzyme having a higher activity than wild type enzyme could be obtained, the weight average molecular weight could be increased, and intracellular accumulation of PHA could be increased.
Also, in The Journal of Biochemistry 133, 139 (2003), an evolutionary engineering modification was applied to the Pseudomonas sp. 61-3 derived polyhydroxyalkanoate synthase gene, which belonged to “the second class”, and Escherichia coli was transformed with this gene together with the genes of β-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB) derived from Ralstonia eutropha, and the transformants were screened for PHB synthetic capability. As the result, it was disclosed that polyhydroxyalkanoate synthase which belonged to the second class could be modified to the synthase having the substrate specificity closed to that of the first class.
Non-Patent Document 1: “Biodegradable plastic handbook”, edited by the biodegradable plastic study group, NTS Inc. P178-197 (1995)
Non-Patent Document 2: APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 68, 2411 (2002)
Non-Patent Document 3: Applied Microbiology and Biotechnology, 59,477 (2002)
Non-Patent Document 4: The Journal of Biochemistry 133, 139 (2003)